Quantitative multiplex detection of nucleic acids

• US 20040146866A1
• Filed: 01/24/2003
• Published: 07/29/2004
• Est. Priority Date: 01/21/2003
• Status: Abandoned Application

• Current Assignee: Guoliang Fu
• Original Assignee: Guoliang Fu
• Inventors: Fu, Guoliang
• Application Number:
  US10/349,780
• Publication Number:
  US 20040146866A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

Methods are provided for quantitative multiplex detection of nucleic acids. Methods of the invention are useful for genotyping mutations, especially single nucleotide polymorphisms (SNPs), for analyzing gene expression profiles, genomic methylation patterns and any specific nucleic acids from any source.

Claims

1. An oligonucleotide primer for the detection of a target nucleic acid sequence, said primer comprising 3′

complementary portion and 5′

non-complementary portion, wherein said 5′

non-complementary portion comprises at least one restriction enzyme site, wherein said restriction site acts as detection marker in a process of detecting said target nucleic acid sequence, whereby a detection signal generated from enzymatic manipulation on said restriction site of a reaction product is indicative of the presence of said target nucleic acid sequence.

2. An oligonucleotide primer of claim 1, wherein said restriction site is allele-specific, gene-specific or SNP-specific.

3. A method of analyzing multiple targets in a polynucleotide, said method comprising

(a) providing a set or sets of multiple primers with target nucleic acids in reactions of primer extension or amplification, wherein said reactions produce nucleic acid products in that each nucleic acid fragment comprises at least one restriction site;

(b) digesting said nucleic acid products on said restriction sites with cognate restriction enzymes;

(c) joining digested products of step (b), whereby randomly joined nucleic acid fragments are created;

(d) amplify joined products of step (c); and

(e) detecting amplified products of step (d).

4. The method of claim 3, wherein said primers are oligonucleotides comprising 3′

complementary portion, or 3′

complementary portion and 5′

non-complementary portion.

5. The method of claim 3, wherein said a set or sets of multiple primer comprise mixtures of target specific primers, wherein primer pairs of forward primers and reverse primers specific for each target are included.

6. The method of claim 3, wherein said a set or sets of multiple primer comprise mixtures of target specific reverse primers and universal primer, wherein said universal primers comprise sequence identical or homologous to said non-complementary portion of target specific forward primers.

7. The method of claim 3, wherein said primers or a subset of said primers comprise capture moiety.

8. The method of claim 3, wherein said capture moiety is biotin.

9. The method of claim 3, wherein said primer extension is first strand cDNA synthesis from target RNA in the presence of a set of target specific primers, random primers or oligo dT primers.

10. The method of claim 3, wherein said primer extension is second strand cDNA synthesis in the presence of a set of target specific primers or random primers.

11. The method of claim 3, wherein said amplification is polymerase chain reaction.

12. The method of claim 3, wherein said amplification is carried out at least once for 1 to 30 cycles.

13. The method of claim 3, wherein said amplification is carried out at least once for 3 to 15 cycles.

14. The method of claim 3, further comprising purification and isolation steps before and/or after said step of digesting said nucleic acid products on said restriction sites with cognate restriction enzymes.

15. The method of claim 14, wherein said purification and isolation steps comprise immobilizing said nucleic acid product on a solid support.

16. The method of claim 15, wherein said solid support is streptavidin coated beads.

17. The method of claim 3, wherein said joining is by ligation using a DNA ligase.

18. The method of claim 3, wherein said amplifying is performed using said a set or sets of multiple primers.

19. The method of claim 3, wherein said amplifying is performed using universal primers having sequences identical or homologous to non-complementary portions of target specific primers.

20. The method of claim 19, wherein said universal primers comprise fluorescence dye labels.

21. The method of claim 3, wherein said restriction sites are located on target sequences or on primer sequences, wherein locations of said restriction sites are chosen such that amplification products digested on said restriction site are distinguishable by their sizes and/or labels.

22. The method of claim 3, wherein said restriction sites are the same restriction site for all nucleic acid fragments generated in said reactiion.

23. The method of claim 3, wherein said restriction sites are different and specific for a subset of targets.

24. The method of claim 3, wherein said detecting is electrophoresis.

25. The method of claim 3, wherein said multiple targets comprise SNPs or mutations.

26. The method of claim 3, wherein said a set or sets of multiple primer comprise mixtures of reverse primers and allele-specific forward primers, wherein two allele-specific forward primers and one common reverse primer for each target are included.

27. The method of claim 26, wherein said allele-specific forward primers comprise 3′

ends which are complementary to either allele at mutation or polymorphism sites.

28. The method of claim 26, wherein each of said two allele-specific forward primers comprises allele-specific restriction site that is different and specific for each allele and is located 5′

of the complementary portion of said allele-specific forward primers.

29. The method of claim 26, wherein said two allele-specific forward primers comprise the same restriction sites that have different locations.

30. The method of claim 26, wherein said allele-specific forward primers comprise first and second restriction sites in non-complementary portion of each primer, wherein said first restriction sites on all forward primers in a set of multiple primers are the same restriction site and are located 5′

of said second restriction sites, whereas said second restriction sites are allele-specific restriction sites which are different and specific for each of said two allele-specific forward primers and are located 5′

of the complementary portion of said allele-specific forward primers.

31. The method of claim 30, wherein said second restriction sites produce 5′

protruding ends after digestion.

32. The method of claims 3, wherein said step of detecting amplified products comprising

(a) purifying amplified products;

(b) digesting amplified products;

(c) extending with a DNA polymerase in the presence of fluorescence dye labeled terminators; and

(d) putting extended DNA product of step (c) into a electrophoresis system.

33. The method of claim 32, wherein said step of purifying amplified products comprises eliminating dNTP and primers.

34. The method of claim 32, wherein said eliminating dNTP and primers comprises incubating with shrimp alkaline phosphatase and exonuclease I.

35. The method of claim 32, wherein said step of digesting amplified products comprises digesting on said restriction sites.

36. The method of claim 32, wherein said step of digesting amplified products comprises digesting on said first restriction sites.

37. The method of claim 32, wherein said step of digesting amplified products comprises digesting on said second restriction sites.

38. The method of claim 32, wherein said terminators are ddNTP.

39. The method of claim 32, wherein said electrophoresis system is a gel or capillary electrophoresis system.

40. The method of claim 32, wherein said electrophoresis system is DNA sequencer.

41. A method of analyzing multiple targets in a polynucleotide, said method comprising

(a) providing different sets of multiple primers with target nucleic acids in separate reactions of primer extension or amplification, wherein said separate reactions produce nucleic acid products in that each nucleic acid fragment comprises at least one restriction site;

(b) digesting said nucleic acid products of said separate reactions on said restriction sites with cognate restriction enzymes;

(c) joining digested products derived from said separate reactions together, whereby randomly joined nucleic acid fragments from said separated reactions are created;

(d) amplify joined products of step (c); and

(e) detecting amplified products of step (d).

42. A kit for use in the analysis and detection of multiple targets in a polynucleotide, said kit comprising:

said a set or sets of multiple primers, said universal primers, said restriction enzymes, said DNA ligase, said DNA polymerase, said ddNTP, buffers for all enzymes, dNTPs.